Endocrine Abstracts

Introduction: Magmas encodes for an integral constituent of the TIM23 translocase complex located in the mitochondrial inner membrane that drives proteins from the intermembrane space into the mitochondrial matrix by functionally interacting with Tim14. We previously demonstrated that Magmas silencing is able to sensitize ACTH-secreting mouse pituitary adenoma cells to pro-apoptotic stimuli, reduce DNA synthesis, accumulate cells in G0/G1 phase with concomitant decrease in S phase, supporting the hypothesis that Magmas may play a role in tumor development by protecting neoplastic cells from apoptosis and by promoting cell proliferation. We then synthesized a protein Magmas inhibitor, reported herein as “Compound 5”, which does not affect viability of cancer cells, but sensitizes them to the pro-apoptotic effects of chemotherapeutic agents such as Staurosporine, Doxorubicin and Cisplatin. These studies provide evidence for a role of Magmas in chemoresistance and indicate that Compound 5 may be useful to restore sensitivity of chemoresistant cancers cells, possibly allowing for a reduction in chemotherapeutic agent effective dose, with consequent decrease in side effects.

Aim: The purpose of this research is to understand whether the mechanism of action of Compound 5 is to disrupt Tim16-Tim14 interaction. This issue is important to explore the key features of Magmas inhibitors, providing information as to the better chemical structure of these compounds which might increase their chemosensitizing effects.

Methods: Tim16 and Tim14 were amplified from pCMV6-Entry vectors and cloned into NanoBiT® Expression Constructs using unique restriction enzyme sites present in the MCS of each vector. The resulting vectors containing Tim16 or Tim14 genes were transformed into One Shot® TOP10 Chemically Competent E. coli and the resulting ligation DNA was miniprepped. For each gene of interest, 4 possible ligation combinations may exist considering both -C and -N terminal tags. These constructs were transfected into chemoresistant cells 2 by 2 (Tim16:Tim14). After 48 h Compound 5 was added at a final concentration of 5uM and after 3 h luminescence from living cells was detected by use of furimazine for up to 2 h.

Results: Tim16 and Tim14 interaction was confirmed by measuring luminescence, developing when Tim16 and Tim14 were -C terminal tagged. The addition of Compound 5, on the other hand, decreased by >65% luciferase emission (P<0,0001).

Discussion: Compound 5 could be further developed to aid the treatment of chemoresistant cancer. Not only it is devoid of toxic activity, but enhances the pro-apoptotic stimuli of chemotherapy, by specifically targeting Tim16-Tim14 interaction.